A Simple Separation Method of the Protein and Polystyrene...

Research ArticleA Simple Separation Method of the Protein and PolystyreneBead-Labeled Protein for Enhancing the Performance ofFluorescent Sensor

Hye Jin Kim1 Dong-Hoon Kang2 Seung-Hoon Yang3 Eunji Lee4 Taewon Ha5

ByungChulLee6YoungbaekKim5KyoSeonHwang1Hyun-JoonShin2 andJinsikKim 4

1Department of Clinical Pharmacology Kyung Hee University Seoul 02447 Republic of Korea2Center for Bionics Korea Institute of Science and Technology Seoul 02792 Republic of Korea3Systems Biotechnology Research Center Korea Institute of Science and Technology Gangneung 25451Republic of Korea4Department of Medical Biotechnology Dongguk University Seoul 04620 Republic of Korea5Center for Nano-Photonics Convergence Technology Korea Institute of Industrial Technology (KITECH)Gwangju 61012 Republic of Korea6Center for BioMicrosystems Korea Institute of Science and Technology (KIST) Seoul 02792Republic of Korea

Correspondence should be addressed to Jinsik Kim lookup2donggukedu

Received 23 March 2018 Revised 31 May 2018 Accepted 7 June 2018 Published 11 July 2018

Academic Editor Subhankar Singha

Copyright copy 2018 Hye Jin Kim et al )is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Dielectrophoresis- (DEP-) based separation method between a protein amyloid beta 42 and polystyrene (PS) beads in differentmicroholes was demonstrated for enhancement of performance for bead-based fluorescent sensor An intensity of nabla|E|2 wasrelative to a diameter of a microhole and the diameters of two microholes for separation between the protein and PS beads weresimulated to 3 μm and 15 μm respectively )e microholes were fabricated by microelectromechanical systems (MEMS) )eseparation between the protein and the PS beads was demonstrated by comparing the average intensity of fluorescence (AIF) byeach molecule Relative AIF was measured in various applying voltage and time conditions and the conditions for allocating thePS beads into 15 μm hole were optimized at 80 mV and 15 min respectively In the optimized condition the relative AIF wasobserved approximately 4908plusmn 0299 Finally in 3 μm and 15 μm hole the AIFs were approximately 3143 and minus1346 by 2 nm ofprotein and about minus2515 and 4211 by 30 nm of the PS beads respectively )e results showed that 2 nm of the protein and 30 nmof PS beads were separated by DEP force in each microhole effectively and that our method is applicable as a newmethod to verifyan efficiency of the labeling for bead-based fluorescent sensor nabla|E|2

1 Introduction

Labeling is one of the essential processes for analyzing andtracking the biomolecules and proceeds by conjugating themolecules with various materials such as isotope markers [1]photochromic compounds [2] and fluorescence polystyrene(PS) beads [3ndash5] Especially fluorescent PS beads havecompetitive price high accessibility and controllability sothat various biomolecules such as protein [6] cell [3] and

deoxyribonucleic acid (DNA) [7] have been conjugated withfluorescent PS beads followed by being quantified andqualified [8ndash10] Qin et al separated the protein whichconjugated with surface-modified fluorescent PS [11] andFakih et al multidetected the viral DNA using goldnanoparticle-coated fluorescent PS beads [12] Howeverfluorescent PS beads are not conjugated with biomoleculesperfectly in other words their labeling efficiency is under100 and consequently not only biomolecules conjugated

HindawiJournal of Analytical Methods in ChemistryVolume 2018 Article ID 8461380 7 pageshttpsdoiorg10115520188461380

with fluorescent PS beads but also nonconjugated bio-molecules existed in the analyte )e nonconjugated bio-molecules decrease the accuracy and sensitivity in analyzingand tracking of the biomolecules hence a method is requiredfor separating the biomolecules conjugated with PS beads andnonconjugated biomolecules namely the residue moleculesSo residue biomolecules after labeling need to be separatedfrom the biomolecules which are conjugated with labelsideally Although centrifugation approaches [13 14] andfluidic-based approaches [15] are suitable for separating theresidue molecules these approaches are complex and requirean additional process

Dielectrophoresis (DEP) resulting from inhomogeneouselectric fields has been utilized for the specific manipulationof the particles cells and viruses as well as biomoleculessuch as DNA and even single protein because of its sim-plicity efficiency and usability [16ndash18] )e intensity of theDEP force is dominated by the size of the molecules andthe strength of the electric fields which occurred betweenthe electrodes so that various molecules are affected bya different intensity of the force according to the size of themolecules and the structure of the electrode Lapizco-Encinas et al concentrated and separated the live anddead bacteria with insulator-based DEP (iDEP) [19] But twotypes of bacteria were separated according to different types ofDEP force negative DEP and positive DEP and Chen et alsuggested a simplified dielectrophoretic-based microfluid de-vice for particle separation [20] But these approaches werelimited for observing the various molecules simultaneously orconsisted of the complex structure

Here we suggest a simple method to separate the non-conjugated protein namely the residue protein and theprotein conjugated with PS beads with the DEP force which isapplicable for verifying the efficiency of labeling betweenprotein and PS beads )e protein and PS beads were sep-arated into two microholes with different diameters andformed on a single electrode according to the intensity of DEPforce induced )e intensity of the DEP force increased whenthe diameter of the microhole was smaller and thus strongerrepulsive force and attractive force occurred in the smallmicrohole than the large one Consequently the smallermolecules residue protein were allocated into the smallmicrohole whereas the bigger molecules PS beads wereexpelled from that small microhole followed by allocating intothe large microhole it means that the protein and PS beadswere separated To verify the separation between protein andPS beads approximately 2 nm of the protein amyloid beta 42and 30nm fluorescent PS beads were used )e diameter ofthe two microholes for separating the protein and PS beadswas optimized to 3 μm and 15 μm respectively by calculatingthe intensity of nabla|E|2 in each microhole with COMSOLsimulation Also an applied voltage to induce the DEP forcewas optimized to 80mV which induced a difference in thenabla|E|2 force approximately 9059-fold between two micro-holes )e microholes were fabricated by micro-electromechanical systems (MEMS) technique andseparation between the protein and polystyrene beads wasdemonstrated by comparing a relative averaged intensity offluorescence (AIF) by each protein and each PS bead

2 Materials and Methods

21 7eory )e molecule present in an inhomogeneouselectric field E is influenced by the DEP force FDEP whichis expressed as follows [21]

FDEP 2πr3εmK(ω) middot nabla| Erarr

|2 (1)

where r εm and K(ω) represent the radius of molecules theeffective permittivity of liquid and the ClausiusndashMossottifactor respectively )e E and gradient of the electric fieldnabla|E|2 are described as follows

E

minusΔV

Δd (2)

nabla|E

| z2V

zd2x

1113954x +z2V

zd2y

1113954y +z2V

zd2z

1113954z (3)

where V and d are the applying voltage and the distancebetween the electrodes respectively On the basis of (1) and(3) the intensity of the DEP force can be modified through(4) as follows

FDEPpropr3

d2(4)

)e intensity of nabla|E|2 is calculated with a finite-elementmodel (FEM) in the ACDC module of COMSOL Multi-physics software 52 (COMSOL Inc USA)

22 Materials )irty nanometres of the carboxylate-modified polystyrene (PS) bead labeled with fluorescence(Sigma-Aldrich Inc Korea) and 2 nm of the TAMRA-labeled beta-amyloid (1ndash42) protein (AnaSpec Inc USA)were used to verify the separation of molecules whoseexcitationemission wavelength (λexλem) was sim470505 nmand sim544572 nm respectively )e protein and PS beadswere diluted with 1mM PBS buffer (Corning Korea Co LtdKorea) to create a 1 ngmiddotmLminus1 solution

23 Fabrication of Microholes Microholes were fabricatedby a standard MEMS process First an insulation layer300 nm of SiO2 and an electrode layer 30 nm of tantalum(Ta) and 150 nm of platinum (Pt) were sequentially de-posited on the 4-inch silicon (Si) wafer by thermal oxidationand sputtering respectively Next an AZ GXR 601 photo-resist (AZ Electronic Materials Luxembourg) was coated bya spin coater (30 s 3000 rpm) and exposed (38 s12mWmiddotcmminus2) )en the hole patterns were etched by in-ductively coupled plasma etching (Oxford Instruments) andthe photoresist was stripped by Microwave Plasma Asher(Plasma-Finish Germany)

24 System Setup for Molecules Separation and FluorescenceAnalysis A DG4062 Series waveform generator (RigolTechnologies Inc USA) (frequency range up to 60MHzvoltage range up to 10V) which applies a sinusoidal ACvoltage for inducing the DEP force in the microhole on

2 Journal of Analytical Methods in Chemistry

a single electrode was used )e intensity of fluorescencewas observed via an electron-multiplying charge-coupleddevice (ANDORiXonEM) an oil immersion 100x lens(Nikon Corp Japan) (NA 14) and an Eclipse Ti invertedmicroscope (Nikon Corp) equipped with a halogen lampand a 593 nm (bandwidth 40 nm) filter and was analyzed byImage-Pro Plus 60 (Media Cybernetics Inc USA) )eaverage intensity of fluorescence (AIF) was calculated byvalues measured at five random positions in the microholeand a value of a relative AIF was calculated by dividing theAIF values measured at each condition by the value in thereference condition

3 Results and Discussion

Protein nonconjugated with PS beads after labeling doesnot emit the fluorescence but binds to the receptor specif-ically so that it impedes the specific binding between theideally conjugated protein with PS beads and receptorfollowed by decreasing the accuracy and reliability in themoleculesrsquo analyzing and tracking process (Figure 1(a)))us the nonconjugated protein namely the residueprotein should be separated from the protein conjugatedwith PS beads When alternating current (AC) voltage isapplied to the electrode with microholes the protein isallocated into each microhole according to the intensity ofthe DEP force that occurred in each microhole)e intensityof the DEP force is related to the diameter of the moleculesand the distance between the electrodes namely the size ofmicroholes as described in (4) and consequently allocatesdifferent molecules into each microhole respectively

residue protein smaller than the PS beads is allocated intothe small microhole whereas the PS beads are placed in thelarge microholemdashtwo molecules separate into small andlarge microholes respectively (Figure 1(b))

In order to separate the residue protein and conjugatedprotein with PS beads in each microhole 45 kDa of amyloidbeta whose diameter was calculated to be approximately2 nm was used as a residue protein and the conjugatedprotein with PS beads was simplified to just PS beads )elength and width of the electrode were fixed to 27 μm and21 μm respectively and the diameter of the small microholed and pitch between two microholes p were fixed to 3 μm)e intensity of the applied AC voltage and size ofmicroholes were optimized via the COMSOL simulation(Figure 2(a)) Firstly maximum intensity of nabla|E|2 in thesmall microhole was simulated according to the applied ACvoltage (Figure 2(b)) Black line and scatter showed themaximum intensity of nabla|E|2 that occurred in the smallmicrohole and red line and scatter indicated the size ofprotein which was allocated into the small microholedepending on the intensity of the applied AC voltage )eintensity of nabla|E|2 increased parabolically and size of theprotein decreased accordingly )e results signified thatapproximately 30mV voltage which resulted in nabla|E|2 withintensity approximately 2310times1013 V2middotmminus3 was required toplace 2 nm of the protein in the 3 μm hole Also themaximum intensity of nabla|E|2 that occurred in the othermicrohole was simulated according to the size of the othermicrohole at the condition that applied 30mV AC voltage(Figure 2(c)) )e diameter of the other microhole wasexpressed as a ratio to the diameter for the 3 μmhole and the

Receptor

Reaction region

Output signalby specific binding

No signalby specific binding

Decreasing the accuracyand reliability

Protein conjugatedwith polystyrene (PS) bead Residue

protein

(a)

DEP force

Residueproteins

Proteins conjugatedwith polystyrene (PS) bead

Smallmicrohole

Largemicrohole

Electrode

(b)

Figure 1 Schematic illustration of a simple separation method of the protein and protein conjugated with polystyrene (PS) beads (a)Intensity of fluorescence by a specific binding of the protein conjugated with PS beads decreased due to a specific binding of the non-conjugated protein expressed as residue protein (b) Residue protein and protein conjugated with PS beads were separated by thedielectrophoresis (DEP) force in different microholes respectively

Journal of Analytical Methods in Chemistry 3

intensity of nabla|E|2 decreased according to the increase in theratio of the diameter In the 6 μmmicrohole (the ratio was 2)intensity of nabla|E|2 was about 0861times 1013 V2middotmminus3 and it is too

strong to allocate 30 nm of the PS beads into the hole nabla|E|2

was approximately 0528times1013 V2middotmminus3 0347times1013 V2middotmminus30255times1013V2middotmminus3 and 0194times1013 V2middotmminus3 in each value of

Length of the electrode

Large microhole

Smallmicrohole

d p

p

Wid

th o

f the

elec

trode

231 times 1013

2

15

1

05

274 times 106

(times1013)

(a)

Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 25

20

15

10

5

0

20 40 60 80 100Applied AC voltage (mV)

10

8

6

4

2

0

Mol

ecul

ar w

eigh

t (kD

a)

(b)

2 3 4 5 6Ratio of diameter to 3 μm microhole (au)

Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 10

08

06

04

02

50

45

40

35

30

25

Dia

met

er o

f PS

base

d (n

m)

(c)

Figure 2 Simulation for separating the molecules by DEP force in the two microholes (a) Distribution of nabla|E|2 at the top view of theelectrode )e diameter of the small microhole and pitch between two microholes were represented as ldquodrdquo and ldquoprdquo respectively(b) According to the applied voltage maximum intensity of nabla|E|2 occurring in the 3 μm hole increased whereas molecular weight of theprotein allocated into the microhole decreased (c) Maximum intensity of nabla|E|2 in the other microhole and the diameter of PS beadsallocated into the hole decreased according to the increase of the diameter

(1) Deposition Tantalumplatinum(TaPt)

Photoresist(PR)

Small microhole

Large microhole

(2) Coating and patterning

(3) Etching and stripping

Siliconsilicondioxide SiSiO2

(a)

Single electrodewith microhole

301 μm

301 μm

301 μm 292 μm

1508 μm

296 μm10 μm

10 μm

(b)

Figure 3 Fabrication of the single electrode consisting of two different-sized microholes by MEMS technology (a) Schematic illustration ofthe fabrication process of the microholes (b) Microscopic image of the two different microholes


the ratio and an optimized size of the microhole for placing30 nm of the PS beads into the hole was verified to be 15 μm)us the two microholes for separating the residue proteinand protein conjugated with PS beads were optimized to3 μm and 15 μm respectively whose difference in the in-tensity was approximately 9059-fold

Two microholes in the electrode were produced viaa standard MEMS process on a 4-inch silicon (Si) wafer )efabrication process consisted of 3 steps (Figure 3(a)) anddetails of the process are described in Material andMethods)e diameter of the fabricated small and large microholeswas approximately 3 μm and 15 μm respectively and pitchbetween two microholes was about 3 μm (Figure 3(b))

Various conditions of DEP intensity of the DEP forceand applied time were optimized by measuring the relativeAIF resulting from placing the PS beads in the 15 μmhole Inorder to optimize the intensity of the DEP force the appliedfrequency required for the DEP force to occur was fixed at50MHz Firstly relative AIF was measured in various ap-plying voltage conditions ranging from 0V (ref) to 500mVand consequently it was maximized at 80mV (Figure 4(a)))e values were approximately 1 1265 1655 1396 and11604 in ref 50 mV 80 mV 100 mV and 500 mV re-spectively )e results indicated that the PS beads were mosteffectively placed in the microhole by the DEP force inducedby the applied voltage 80mV consequently the intensity ofthe applied voltage was settled to 80mV Also in order tooptimize the applied time condition of DEP force the

relative AIF wasmeasured according to time every 3minutesup to 21 minutes (Figure 4(b)) )e AIF increased graduallydepending on the time up to 15min and was saturatedafterward each value of the AIF was approximately 09071048 1184 1450 1526 1535 and 1536 according to theapplied time Hence the applied voltage and time wereoptimized to 80mV and 15 minutes respectively and therelative AIF was observed to be approximately 4908plusmn 0299in the optimized condition (Figure 4(c)) )e result dem-onstrated that the PS beads were allocated into the 15 μmholeeffectively

Relat

ive A

IF (a

u)

16

14

12

10

Ref 50 80 100 500Applied voltage (mV)

(a)

Relat

ive A

IF (a

u)

3 6 9 12 15 18 21Applied time (min)

15

10

05

(b)

6

4

2

0Ref condition Optimized condition

Relat

ive A

IF (a

u)

(c)

Figure 4 Optimization of the DEP condition by measuring the average intensity of fluorescence (AIF) of the PS beads in the 15 μm holeRelative AIF was verified according to (a) the applied voltage and (b) the applied time of AC voltage (c) Relative AIF by the PS beads in the15 μm hole was compared in each reference and optimized DEP condition

6

4

2

0

ndash2

ndash4

Rela

tive A

IF

3 microm hole 15 microm hole

2 nm protein30 nm polystyrene bead

Figure 5 Relative AIF by 2 nm of the protein and 30 nm of the PSbeads in 3 μm and 15 μm holes respectively


Finally based on these results a separation of the proteinand PS beads in 3μm and 15μm holes respectively wasdemonstrated (Figure (5)) It was also confirmed by comparingthe AIF by eachmolecule in the twomicroholes at the previousoptimized condition)e relative AIF by 2nm of the protein inthe 3μmhole was a positive value but the value by 30nmof thePS beads was negative and the values were approximately 3143and minus1346 respectively whereas in the 15μmhole the relativeAIFs by the protein and the PS beads showed an opposite signcompared with the previous values and the values were ap-proximately minus2515 and 4211 respectively )e negative valueof the AIF indicated that the molecules were moving far awayowing to the strong DEP force in the microhole and thepositive value of the AIF signified that the molecules wereattracted and trapped into the microhole by the DEP force)us the results signified that the DEP force allocated 2nm ofthe protein and 30nm of the PS beads into 3μm and 15μmholes respectively )e results demonstrated that 2nm of theprotein and 30nmof the PS beads were separated byDEP forcein each microhole effectively

4 Conclusions

In this paper a simple method for separation between 2 nmof the protein and PS beads into different microholes re-spectively by the DEP force was demonstrated In order toseparate twomolecules the diameter of the twomicroholes wassimulated and the intensity of the DEP force induced in themicroholes was calculated via simulation )e optimized di-ameter of the two microholes was 3μm and 15μm anda difference in the DEP force between two microholes wasapproximately 9059-fold )e condition of the DEP force toseparate twomolecules was optimized experimentally intensityof the AC voltage was 80mV and the applied time was 15minutes)emolecules which were separated by the DEP forcein each microhole were verified by measuring the relative AIFby each molecule In 3μm and 15μm holes the AIFs wereapproximately 3143 and minus1346 by 2nm of the protein andabout minus2515 and 4211 by 30nm of the PS beads respectivelyConsequently the results demonstrated that 2nm of theprotein and 30nmof the PS beads were separated byDEP forcein each microhole effectively Our method has high expand-ability in separation of various-sized molecules and further-more it is applicable for verification of the labeling efficiency

Data Availability

)e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

)e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

)is work was mainly supported by the Dongguk UniversityResearch Fund of 2017 Taewon Ha and Youngbaek Kim are

grateful for financial support from the Korea Institute ofIndustrial Technology (Project no EO170047)

References

[1] X Chen L M Smith and E M Bradbury ldquoSite-specific masstagging with stable isotopes in proteins for accurate and ef-ficient protein identificationrdquo Analytical Chemistry vol 72no 6 pp 1134ndash1143 2000

[2] M Lummer F Humpert M Wiedenlubbert M SauerM Schuttpelz and D Staiger ldquoA new set of reversiblyphotoswitchable fluorescent proteins for use in transgenicplantsrdquo Molecular Plant vol 6 no 5 pp 1518ndash1530 2013

[3] H Sahoo ldquoFluorescent labeling techniques in biomoleculesa flashbackrdquo RSC Advances vol 2 no 18 pp 7017ndash70292012

[4] D Jung K Min J Jung W Jang and Y Kwon ldquoChemicalbiology-based approaches on fluorescent labeling of proteinsin live cellsrdquoMolecular BioSystems vol 9 no 5 pp 862ndash8722013

[5] M M Bonar and J C Tilton ldquoHigh sensitivity detection andsorting of infectious human immunodeficiency virus (HIV-1)particles by flow virometryrdquo Virology vol 505 pp 80ndash902017

[6] C Obermaier A Griebel and R Westermeier ldquoPrinciples ofprotein labeling techniquesrdquo Proteomic Profiling Methodsand Protocols vol 1295 pp 153ndash165 2015

[7] T T Weil R M Parton and I Davis ldquoMaking the messageclear visualizing mRNA localizationrdquo Trends in Cell Biologyvol 20 no 7 pp 380ndash390 2010

[8] J Lu G Getz E A Miska et al ldquoMicroRNA expressionprofiles classify human cancersrdquo Nature vol 435 pp 834ndash838 2005

[9] B S Edwards T Oprea E R Prossnitz and L A Sklar ldquoFlowcytometry for high-throughput high-content screeningrdquoCurrent Opinion in Chemical Biology vol 8 no 4 pp 392ndash398 2004

[10] X H Gao and S M Nie ldquoQuantum dot-encoded mesoporousbeads with high brightness and uniformity rapid readoutusing flow cytometryrdquo Analytical Chemistry vol 76 no 8pp 2406ndash2410 2004

[11] L Qin X W He W Zhang W Y Li and Y K ZhangldquoSurface-modified polystyrene beads as photograftingimprinted polymer matrix for chromatographic separation ofproteinsrdquo Journal of Chromatography A vol 1216 no 5pp 807ndash814 2009

[12] H H Fakih M M Itani and P Karam ldquoGold nanoparticles-coated polystyrene beads for the multiplex detection of viralDNArdquo Sensors and Actuators B Chemical vol 250pp 446ndash452 2017

[13] E Fernandez-Vizarra M J Lopez-Perez and J A EnriquezldquoIsolation of biogenetically competent mitochondria frommammalian tissues and cultured cellsrdquoMethods vol 26 no 4pp 292ndash297 2002

[14] U Michelsen and J von Hagen ldquoIsolation of subcellularorganelles and structuresrdquo in Methods in Enzymology pp305mdash328 Academic Press Cambridge MA USA 2009

[15] A A S Bhagat H Bow H W Hou S J Tan J Han andC T Lim ldquoMicrofluidics for cell separationrdquo Medical andBiological Engineering and Computing vol 48 pp 999ndash10142010

[16] S Paracha and C Hestekin ldquoField amplified sample stackingof amyloid beta (1-42) oligomers using capillary electro-phoresisrdquo Biomicrofluidics vol 10 no 3 article 033105 2016


[17] T D Mai F Qukacine and M Taverna ldquoMultiple capillaryisotachophoresis with repetitive hydrodynamic injections forperformance improvement of the electromigration pre-concentrationrdquo Journal of Chromatography A vol 1453pp 116ndash123 2016

[18] L Zheng J P Brody and P J Burke ldquoElectronic manipulationof DNA proteins and nanoparticles for potential circuitassemblyrdquo Biosensors and Bioelectronics vol 20 no 3pp 606ndash619 2004

[19] B H Lapizco-Encinas B A Simmons E B Cummings andY Fintschenko ldquoDielectrophoretic concentration and sepa-ration of live and dead bacteria in an array of insulatorsrdquoAnalytical Chemistry vol 76 no 16 pp 1571ndash1579 2004

[20] X Chen Y Ren W Liu et al ldquoA simplified microfluidicdevice for particle separation with two consecutive stepsinduced charge electro-osmotic prefocusing and dielec-trophoretic separationrdquo Analytical Chemistry vol 89 no 17pp 9583ndash9592 2017

[21] A Ramos H Morgan N G Green and A Castellanos ldquoAcelectrokinetics a review of forces in microelectrode struc-turesrdquo Journal of Physics D Applied Physic vol 31 no 18p 2338 1998


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Submit your manuscripts atwwwhindawicom

with fluorescent PS beads but also nonconjugated bio-molecules existed in the analyte )e nonconjugated bio-molecules decrease the accuracy and sensitivity in analyzingand tracking of the biomolecules hence a method is requiredfor separating the biomolecules conjugated with PS beads andnonconjugated biomolecules namely the residue moleculesSo residue biomolecules after labeling need to be separatedfrom the biomolecules which are conjugated with labelsideally Although centrifugation approaches [13 14] andfluidic-based approaches [15] are suitable for separating theresidue molecules these approaches are complex and requirean additional process

Dielectrophoresis (DEP) resulting from inhomogeneouselectric fields has been utilized for the specific manipulationof the particles cells and viruses as well as biomoleculessuch as DNA and even single protein because of its sim-plicity efficiency and usability [16ndash18] )e intensity of theDEP force is dominated by the size of the molecules andthe strength of the electric fields which occurred betweenthe electrodes so that various molecules are affected bya different intensity of the force according to the size of themolecules and the structure of the electrode Lapizco-Encinas et al concentrated and separated the live anddead bacteria with insulator-based DEP (iDEP) [19] But twotypes of bacteria were separated according to different types ofDEP force negative DEP and positive DEP and Chen et alsuggested a simplified dielectrophoretic-based microfluid de-vice for particle separation [20] But these approaches werelimited for observing the various molecules simultaneously orconsisted of the complex structure

Here we suggest a simple method to separate the non-conjugated protein namely the residue protein and theprotein conjugated with PS beads with the DEP force which isapplicable for verifying the efficiency of labeling betweenprotein and PS beads )e protein and PS beads were sep-arated into two microholes with different diameters andformed on a single electrode according to the intensity of DEPforce induced )e intensity of the DEP force increased whenthe diameter of the microhole was smaller and thus strongerrepulsive force and attractive force occurred in the smallmicrohole than the large one Consequently the smallermolecules residue protein were allocated into the smallmicrohole whereas the bigger molecules PS beads wereexpelled from that small microhole followed by allocating intothe large microhole it means that the protein and PS beadswere separated To verify the separation between protein andPS beads approximately 2 nm of the protein amyloid beta 42and 30nm fluorescent PS beads were used )e diameter ofthe two microholes for separating the protein and PS beadswas optimized to 3 μm and 15 μm respectively by calculatingthe intensity of nabla|E|2 in each microhole with COMSOLsimulation Also an applied voltage to induce the DEP forcewas optimized to 80mV which induced a difference in thenabla|E|2 force approximately 9059-fold between two micro-holes )e microholes were fabricated by micro-electromechanical systems (MEMS) technique andseparation between the protein and polystyrene beads wasdemonstrated by comparing a relative averaged intensity offluorescence (AIF) by each protein and each PS bead

2 Materials and Methods

21 7eory )e molecule present in an inhomogeneouselectric field E is influenced by the DEP force FDEP whichis expressed as follows [21]

FDEP 2πr3εmK(ω) middot nabla| Erarr

|2 (1)

where r εm and K(ω) represent the radius of molecules theeffective permittivity of liquid and the ClausiusndashMossottifactor respectively )e E and gradient of the electric fieldnabla|E|2 are described as follows

E

minusΔV

Δd (2)

nabla|E

| z2V

zd2x

1113954x +z2V

zd2y

1113954y +z2V

zd2z

1113954z (3)

where V and d are the applying voltage and the distancebetween the electrodes respectively On the basis of (1) and(3) the intensity of the DEP force can be modified through(4) as follows

FDEPpropr3

d2(4)

)e intensity of nabla|E|2 is calculated with a finite-elementmodel (FEM) in the ACDC module of COMSOL Multi-physics software 52 (COMSOL Inc USA)

22 Materials )irty nanometres of the carboxylate-modified polystyrene (PS) bead labeled with fluorescence(Sigma-Aldrich Inc Korea) and 2 nm of the TAMRA-labeled beta-amyloid (1ndash42) protein (AnaSpec Inc USA)were used to verify the separation of molecules whoseexcitationemission wavelength (λexλem) was sim470505 nmand sim544572 nm respectively )e protein and PS beadswere diluted with 1mM PBS buffer (Corning Korea Co LtdKorea) to create a 1 ngmiddotmLminus1 solution

23 Fabrication of Microholes Microholes were fabricatedby a standard MEMS process First an insulation layer300 nm of SiO2 and an electrode layer 30 nm of tantalum(Ta) and 150 nm of platinum (Pt) were sequentially de-posited on the 4-inch silicon (Si) wafer by thermal oxidationand sputtering respectively Next an AZ GXR 601 photo-resist (AZ Electronic Materials Luxembourg) was coated bya spin coater (30 s 3000 rpm) and exposed (38 s12mWmiddotcmminus2) )en the hole patterns were etched by in-ductively coupled plasma etching (Oxford Instruments) andthe photoresist was stripped by Microwave Plasma Asher(Plasma-Finish Germany)

24 System Setup for Molecules Separation and FluorescenceAnalysis A DG4062 Series waveform generator (RigolTechnologies Inc USA) (frequency range up to 60MHzvoltage range up to 10V) which applies a sinusoidal ACvoltage for inducing the DEP force in the microhole on







Receptor

Reaction region





protein

(a)

DEP force

Residueproteins


Smallmicrohole

Largemicrohole

Electrode

(b)







Large microhole

Smallmicrohole

d p

p

Wid

th o

f the

elec

trode

231 times 1013

2

15

1

05

274 times 106

(times1013)

(a)

Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 25

20

15

10

5

0


10

8

6

4

2

0

Mol

ecul

ar w

eigh

t (kD

a)

(b)


Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 10

08

06

04

02

50

45

40

35

30

25

Dia

met

er o

f PS

base

d (n

m)

(c)



Photoresist(PR)

Small microhole

Large microhole




(a)


301 μm

301 μm

301 μm 292 μm

1508 μm

296 μm10 μm

10 μm

(b)







Relat

ive A

IF (a

u)

16

14

12

10


(a)

Relat

ive A

IF (a

u)


15

10

05

(b)

6

4

2


Relat

ive A

IF (a

u)

(c)


6

4

2

0

ndash2

ndash4

Rela

tive A

IF






4 Conclusions


Data Availability




Acknowledgments



References





























Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls









Receptor

Reaction region





protein

(a)

DEP force

Residueproteins


Smallmicrohole

Largemicrohole

Electrode

(b)







Large microhole

Smallmicrohole

d p

p

Wid

th o

f the

elec

trode

231 times 1013

2

15

1

05

274 times 106

(times1013)

(a)

Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 25

20

15

10

5

0


10

8

6

4

2

0

Mol

ecul

ar w

eigh

t (kD

a)

(b)


Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 10

08

06

04

02

50

45

40

35

30

25

Dia

met

er o

f PS

base

d (n

m)

(c)



Photoresist(PR)

Small microhole

Large microhole




(a)


301 μm

301 μm

301 μm 292 μm

1508 μm

296 μm10 μm

10 μm

(b)







Relat

ive A

IF (a

u)

16

14

12

10


(a)

Relat

ive A

IF (a

u)


15

10

05

(b)

6

4

2


Relat

ive A

IF (a

u)

(c)


6

4

2

0

ndash2

ndash4

Rela

tive A

IF






4 Conclusions


Data Availability




Acknowledgments



References





























Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








Large microhole

Smallmicrohole

d p

p

Wid

th o

f the

elec

trode

231 times 1013

2

15

1

05

274 times 106

(times1013)

(a)

Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 25

20

15

10

5

0


10

8

6

4

2

0

Mol

ecul

ar w

eigh

t (kD

a)

(b)


Max

imum

nabla|E

|2 (1013

V2 middotm

ndash3) 10

08

06

04

02

50

45

40

35

30

25

Dia

met

er o

f PS

base

d (n

m)

(c)



Photoresist(PR)

Small microhole

Large microhole




(a)


301 μm

301 μm

301 μm 292 μm

1508 μm

296 μm10 μm

10 μm

(b)







Relat

ive A

IF (a

u)

16

14

12

10


(a)

Relat

ive A

IF (a

u)


15

10

05

(b)

6

4

2


Relat

ive A

IF (a

u)

(c)


6

4

2

0

ndash2

ndash4

Rela

tive A

IF






4 Conclusions


Data Availability




Acknowledgments



References





























Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








Relat

ive A

IF (a

u)

16

14

12

10


(a)

Relat

ive A

IF (a

u)


15

10

05

(b)

6

4

2


Relat

ive A

IF (a

u)

(c)


6

4

2

0

ndash2

ndash4

Rela

tive A

IF






4 Conclusions


Data Availability




Acknowledgments



References





























Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





4 Conclusions


Data Availability




Acknowledgments



References





























Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls















Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




A Simple Separation Method of the Protein and Polystyrene...

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Transcript of A Simple Separation Method of the Protein and Polystyrene...